Method for detecting pathological pregnancy

ABSTRACT

A method for the detection of the presence of inflammation in a patient by measuring the amount of circulating intercellular adhesion molecule (cICAM-1) in a sample of one or more bodily fluids of the patient and then comparing the amount of cICAM-1 in the sample to standards normal for the bodily fluid or fluids assayed. The amount of cICAM-1 can be measured using anti-ICAM-1 antibodies. Higher than normal amounts of cICAM-1 indicate the presence of inflammation.

This is a division of application Ser. No. 695,173, filed May 3, 1991,now U.S. Pat. No. 5,223,396.

FIELD OF THE INVENTION

This invention relates to a method for detecting inflammation in apatient by measuring the amount of circulating intercellular adhesionmolecule-1 (cICAM-1) in bodily fluids of the patient. In particular,this invention relates a method for detecting inflammation in a patientby measuring the amount of cICAM-1 in bodily fluids of the patient usingantibodies specific for intercellular adhesion molecule-1 (ICAM-1).

BACKGROUND OF THE INVENTION

Intercellular adhesion molecule-1 (ICAM-1) is a cytokine-inducibleadhesion molecule expressed on cells of multiple lineages at sites ofinflammation. See, e.g., Vejlsgaard et al, J. Amer. Acad. Demtol. 20:782 (1989) and Sobel et al, Am. J. Pathol. 136: 1309 (1990). It is aligand for at least 2 members of the CD18 family of leukocyte adhesionmolecules (LFA-1 and Mac-1) and mediates, in part, granulocyteextravasation, lymphocyte mediated cytotoxicity and the development ofspecific immunological responses involving cell-cell interactions. See,e.g., Springer, T. A., Nature 346: 425 (1990). Antibodies to ICAM-1 havebeen shown to inhibit leukocyte adhesion to endothelial cells,granulocyte migration through endothelium, mitogen and antigen inducedlymphocyte proliferation and mixed lymphocyte reactions in vitro. See,e.g., Smith et al, J. Clin. Invest. 82: 813 (1987). In vivo, antibodiesto ICAM-1 inhibit neutrophil trafficking into inflamed lungs in rabbits,nonhuman primate kidney and heart allograft refection, and antigeninduced airway eosinophil influx and airway hyperresponsiveness. See,e.g., Barton et al, J. Immunol. 143: 1278 (1989). Structurally, ICAM-1is a member of the immunoglobulin supergene family with 5immunoglobulin-like domains, a single transmembrane region and a shortcytoplasmic tail. See, e.g., Staunton et al, Cell 52: 925 (1988). ICAM-1has been identified as a receptor for the major rhinovirus group and agenetically engineered form of ICAM-1 lacking the cytoplasmic tail andtransmembrane region has been shown to inhibit rhinovirus infection invitro. Marlin et al, Nature 344: 70 (1990) (hereinafter referred to as"Marlin et al"), herein incorporated by reference.

It is the purpose of this invention to provide a method for thedetection of inflammation in a patient by measuring the amount of asoluble form of ICAM-1 in circulation (cICAM-1) in the bodily fluids ofthe patient.

SUMMARY OF THE INVENTION

This invention relates to a method for detecting the presence ofinflammation in a patient which comprises measuring the amount ofcICAM-1 in a sample of one or more bodily fluids of the patient and thencomparing the amount of cICAM-1 in the sample to standards normal forthe bodily fluid or fluids assayed. Higher than normal amounts ofcICAM-1 indicate the presence of inflammation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is schematic representation of a sandwich assay for cICAM-1 usingan immobilized anti-ICAM-1 antibody directed against domain 4 of ICAM-1and a labelled anti-ICAM-1 antibody directed against domain 1 of ICAM-1.

FIG. 2 is a graphic representation of cICAM-1 levels in sera frompatients with normal serum, LAD patients and patients with Kawasakisdisease.

FIG. 3 is a graphic representation of cICAM-1 levels in the amnioticfluid of pregnant women at 16 weeks gestation, pregnant women who had apreterm spontaneous delivery, pregnant women who had preterm delivery byc-section, pregnant women who had a delivery at term by c-section andpregnant women who had a normal delivery at term.

FIG. 4 is a graphic representation of cICAM-1 levels in synovial fluidof patients with rheumatoid arthritis (RA), osteoarthritis (OA),psoriatic arthritis, gouty arthritis, and chondrocalciosis.

FIGS. 5A and 5B are a graphic representation of cICAM-1 levels in thebile and plasma of a patient rejecting a liver transplant and anon-rejecting patient, over a period of time from 2 days post-transplantto 15 days post-transplant.

FIG. 6 is a graphic representation of cICAM-1 levels in the bile ofliver transplant patients who rejected the transplant, who hadcomplications, and who were stable.

DETAILED DESCRIPTION OF THE INVENTION

The term "circulating ICAM-1" or "cICAM-1", for the purposes of thisinvention, means the soluble form of ICAM-1 in circulation in the bodilyfluids of a patient including, for example, serum, bile, synovial fluid,and amniotic fluid. Most monoclonal antibodies that bind to ICAM-1 bindto cICAM-1. Additionally, immobilized cICAM-1 binds to LFA-1 bearingcells, demonstrating that cICAM-1 is structurally similar to geneticallyengineered sICAM-1 (Marlin et al). The apparent molecular weight ofcICAM-1 is approximately 150 kD, as measured by size exclusionchromatography. sICAM-1, as prepared in Marlin et al, has a similarapparent molecular weight.

The discovery of cICAM-1 in bodily fluids and the observation thathigher than normal levels of cICAM-1 in the bodily fluids of a patientare indicative of the presence of inflammation, is the basis for theassays useful in the method of this invention.

The term "inflammation", as used herein, is meant to include both thereactions of the specific defense system, and the reactions of thenon-specific defense system.

As used herein, the term "specific defense system" is intended to referto that component of the immune system that reacts to the presence ofspecific antigens. Inflammation is said to result from a response of thespecific defense system if the inflammation is caused by, mediated by,or associated with a reaction of the specific defense system. Examplesof inflammation resulting from a response of the specific defense systeminclude the response to antigens such as rubella virus, autoimmunediseases such as lupus erythematosus, rheumatoid arthritis, Reynaud'ssyndrome, multiple sclerosis etc., delayed type hypersensitivityresponse mediated by T-cells, etc. Chronic inflammatory diseases and therejection of transplanted tissue and organs are further examples ofinflammatory reactions of the specific defense system.

As used herein, a reaction of the "non-specific defense system" isintended to refer to a reaction mediated by leukocytes incapable ofimmunological memory. Such cells include granulocytes and macrophages.As used herein, inflammation is said to result from a response of thenon-specific defense system, if the inflammation is caused by, mediatedby, or associated with a reaction of the non-specific defense system.Examples of inflammation which result, at least in part, from a reactionof the non-specific defense system include inflammation associated withconditions such as: adult respiratory distress syndrome (ARDS) ormultiple organ injury syndromes secondary to septicemia or trauma;reperfusion injury of myocardial or other tissues; acuteglomerulonephritis; reactive arthritis; dermatoses with acuteinflammatory components; acute purulent meningitis or other centralnervous system inflammatory disorders; thermal injury; hemodialysis;leukophoresis; ulcerative colitis; Crohn's disease; necrotizingenterocolitis; granulocyte transfusion associated syndromes; andcytokine-induced toxicity.

The selection of bodily fluids to be assayed will vary dependingprimarily on the type of inflammation to be detected. The bodily fluidor fluids selected should be either in contact with or produced at, thesite of the inflammation. For the purposes of the method this invention,the bodily fluids should be substantially free of cells. For example,the presence of inflammation (and accordingly, elevated levels ofcICAM-1) in synovial fluid may indicate the existence of rheumatoidarthritis. Kawasakis disease results in elevated levels of cICAM-1 inserum of patients afflicted with the disease. In monitoring the progressof liver transplants, elevated levels of cICAM-1 in the bile of apatient who received a liver transplant, indicate rejection of thetransplant. Elevated levels of cICAM-1 in the amniotic fluid of apregnant woman indicate a potential risk of a problem pregnancy.

Preferably, the assay useful in this invention is an immunoassay fordetecting the presence of inflammation in a patient which comprises thesteps of:

a) contacting a sample of one or more bodily fluids of the patient, witha first antibody capable of binding to ICAM-1 and a labelled secondantibody capable of binding to ICAM-1;

b) determining the amount of bound labelled second antibody as a measureof the amount of cICAM-1 in the sample; and

c) comparing the amount of cICAM-1 in the sample with standards ofcICAM-1 normal for the bodily fluid or fluids assayed. Preferably, theantibodies are monoclonal antibodies.

Any immunoassay which produces quantifiable results can be used. Thisincludes competitive and non-competitive binding assays, single site ormulti-site. Preferably, the immunoassay used is a sandwich assay whereinthe first antibody is immobilized and the labelled second antibody issoluble. Sandwich assays are described, for example, in U.S. Pat. Nos.4,376,110 and 4,244,940.

In a more preferred embodiment of this invention, the immunoassay is asandwich assay which comprises the steps of:

a) contacting a sample of one or more bodily fluids of a patient with animmobilized first antibody capable of binding to ICAM-1, the immobilizedfirst antibody being insoluble in the sample, and with a solublelabelled second antibody capable of binding to ICAM-1, to form a finalinsoluble complex of the labelled second antibody, cICAM-1 and theimmobilized first antibody;

b) determining the amount of labelled second antibody bound to the finalinsoluble complex, as a measure of the amount of cICAM-1 in the sample;and

c) comparing the amount of cICAM-1 in the sample with standards ofcICAM-1 normal for the bodily fluid or fluids assayed.

The immobilized first antibody is preferably immobilized on a solidsupport. The solid support can be any of the known support materialsuseful in prior art assays, such as cellulose, agarose, sepharose,polystyrene, nylon, polyacrylamide, latex, glass, magnetizableparticles, nitrocellulose, etc. Preferably, the solid support ispolystyrene. The antibody can be immobilized onto the solid support byany procedure which produces an immobilized antibody capable of bindingto ICAM-1, such as by adsorption or covalent binding. Procedures foraccomplishing such immobilization are well known to the art. Forexample, the antibody can be adsorbed in microtiter wells as describedin Example 1 below and in Erlich et al, Methods in Enzymology 68: 443(1979), or can be immobilized on a solid support using a bifunctionalreagent as described in Kagedal et al, Clinica Chimica Acta 78: 103(1977).

The amount of immobilized first antibody utilized in the method of thisinvention must be sufficient to bind a detectable quantity of cICAM-1.This amount will vary depending upon the type of inflammation to bedetected, antibody, label used, etc., and should be determinedempirically. In general, it is preferred that about 1 μg to about 10 μgof first antibody immobilized on the solid support per 50 μl of fluidsample, be utilized.

The labelled second antibody can be labeled by known means (e.g., withenzymatic, fluorogenic, radiometric, bioluminescent, affinity,chemiluminescent, colorimetric, etc., labels and markers), provided thatthe label does not have a deleterious effect on the binding of theantibody to cICAM-1. Procedures for accomplishing such labelling arewell known to the art. For example, the procedure described in Example 1can be used in biotinylating an antibody for the purposes of thisinvention, or a procedure such as the one described in Woodhead et al,Clinical Chemistry 29(8): 1474 (1983), can be used in labeling anantibody for the purposes of this invention. The amount of labelledsecond antibody utilized in the method of this invention must besufficient to permit the detection of the cICAM-1 bound by the labelledsecond antibody. This amount will vary depending primarily upon the typeof label used and should be determined empirically. Preferably, thesecond antibody carries a label that is capable of effecting a colorchange indicative of the presence of cICAM-1.

The final insoluble complex of labelled second antibody, cICAM-1, andimmobilized first antibody bound to a solid support, can be producedusing three assay procedures, referred to as a forward assay, a reverseassay, and a simultaneous assay. In the forward assay, the sample isfirst incubated with an immobilized first antibody for an appropriateperiod of time to form a first insoluble complex and then the firstinsoluble complex so formed is incubated with a soluble labelled secondantibody for an appropriate period of time, to form the final insolublecomplex. In the forward assay, the immobilized first antibody and thesoluble labelled second antibody should be two different anti-ICAM-1antibodies, which do not interfere with the binding of each other to thecICAM-1 molecule.

In the reverse assay, the sample is first incubated with a solublelabelled second antibody for an appropriate period of time to form asoluble complex of soluble labelled second antibody and cICAM-1. Thesoluble complex so formed is then incubated with an immobilized firstantibody for an appropriate period of time to form the final insolublecomplex. In the reverse assay, the immobilized first antibody and thesoluble labelled second antibody should be two different anti-ICAM-1antibodies which do not interfere with the binding of each other to thecICAM-1 molecule.

In the simultaneous assay, the sample is incubated with an immobilizedfirst antibody and a soluble labelled second antibody at the same timeto form the final insoluble complex. In the simultaneous assay, theimmobilized first antibody and the soluble labelled second antibodyshould be two different anti-ICAM-1 antibodies, which do not interferewith the binding of each other to the cICAM-1 molecule.

The incubation conditions for each of the steps of the forward, reverseand simultaneous assays can vary, depending on time, temperature, andfinal incubation volume, but, preferably, each incubation step should beconducted for at least 15 minutes, more preferably for 30 minutes orlonger, at room temperature or higher, more preferably, at 37° C.

After the incubation, the final insoluble complex is separated from theincubation medium (i.e., sample, unbound soluble labelled secondantibody, etc.). Since the final insoluble complex is insoluble in theincubation medium, it can be separated from the incubation medium byconventional means.

The uptake of the soluble labelled second antibody is directly relatedto the presence of the cICAM-1 bound to the complex. The amount of labelassociated with the final insoluble complex can be determined by atleast two methods: (1) direct quantitation of the label associated withthe final insoluble complex, or (2) indirect quantitation of the labelremaining in the incubation medium after separation and then subtractingthe amount from the total label offered. The appropriate quantitationprocedure will depend largely on the label used.

The anti-ICAM-1 antibodies useful in the method of this invention can bepolyclonal, monoclonal or recombinantly produced. Techniques useful inthe preparation of the anti-ICAM-1 antibodies are well known in the art.See, e.g., Kohler and Milstein, Nature 356: 495 (1975); U.S. Pat. No.4,816,567; Rothlein et al, supra, and European Patent Application SerialNo. 289,949.

Other ligands of ICAM-1, such as LFA-1, may also be used to detect andmeasure cICAM-1 in the bodily fluids of a patient.

The following examples illustrate the method of this invention.

EXAMPLE 1 Detection of cICAM-1 in Human Sera

Sera from 10 normal individuals were compared to sera from 4 patientssuffering from leukocyte adhesion deficiency (LAD) and to sera from 12patients suffering from Kawasakis disease, to determine if there weredetectable amounts of cICAM-1 in serum samples.

A. Preparation of Biologicals

Soluble ICAM-1 (sICAM-1) was prepared as described in Marlin et al;serially diluted in 1% bovine serum (Sigma, St. Louis, Mo.) Dulbecco'sphosphate-buffered saline (Media Tech, Washington, D.C.) (BSA-dPBS); andaliquots frozen at -70° C.

Sera from normal human blood was collected into sterile Vacuutainersfrom drug free donors (10) by venipuncture. Blood was allowed to clotfor at least one-half hour prior to collection of sera. Sera fromleukocyte adhesion deficiency (LAD) patients (4) were obtained from Dr.Donald Anderson of Baylor University, Houston, Tex. Sera from patientswith Kawasakis disease (12) were obtained from Dr. Jane Neuberger andDr. Fred Rosen of the Center for Blood Research, Inc., Boston, Mass.

B. Preparation of Monoclonal Antibodies

Antibodies RR1/1 and R6.5 directed against domains 1 and 2 of ICAM-1were prepared as described in Rothlein et al, J. Immunol. 137: 1270(1986) and European Patent Application Serial No. 289,949, respectively.

Antibody CL203.4, directed against domain 4 of ICAM-1, was provided byDr. Soldano Ferrone of New York Medical College, Valhalla, N.Y.

Antibody CA7, directed against domain 5 of ICAM-1, was prepared asfollows:

BALB/C mice were subcutaneously immunized with 150 μg of sICAM-1 in0.2ml of complete Freunds adjuvant:saline (1:1 emulsion) on day -76; 100μg of sICAM-1 in incomplete Freund's adjuvant:saline (1:1 emulsion) in0.2 ml on day -45. On days -4 and -3, the mice were further immunizedwith 150 μg of sICAM-1 intraperitoneally. A fusion was then performed onday 0 between the spleens of the immunized mice and P3x63Ag8.658 myelomacells using the protocol described in Rothlein et al, J. Immunol. 139:1270 (1986). The resultant hybridoma antibodies were screened by ELISAfor their ability to bind to sICAM-1 on plates. Selected hybridomas weresubcloned twice. CA7 was determined to bind to domain 5 of ICAM-1 byimmunoperoxidase staining of cytocentrifuge preparations of COS cellstransfected with various constructs of ICAM-1 deletion mutants obtainedfrom Dr. Donald Staunton and Dr. Timothy Springer, Center for BloodResearch, Inc., Boston, Mass.

Anti-LFA-1 (anti-CD11a) antibody R3.1 was prepared as described in Smithet al, J. Clin. Invest. 82: 1746 (1988).

Antibodies were biotinylated as described in Guesdon et al, J.Histochem. Cytochem. 27: 1113 (1979).

C. ELISA for cICAM-1

CL203.4 (10 μg/ml in DPBS) was added to each well in 96 well flat bottomE.I.A. microtiter plates (Linbro) at 50 μl/well at room temperature for1 hour. Wells were then washed three times with DPBS and then blockedwith 200 μl of 2% BSA-DPBS for 1 hour at 37° C. Wells were then flickedempty and a titration of sICAM-1 standards (8 to 1024 ng/ml) and serasamples (titrated in 1% BSA-DPBS) were then added (50 μl/well) intriplicate for 1 hour at 37° C. Wells were then washed three times withDPBS. Biotinylated R6.5 was then added to each well, at 2 μg/ml (50μl/well) for 30 minutes at 37° C. Wells were then washed three timeswith DPBS. 50 μl/well of horseradish peroxidase streptavidin (Zymed, SanFrancisco, Calif.) (1:4000) was then added to each well for 30 minutesat 37° C., followed by three washes with DPBS and one wash withsubstrate buffer (Zymed). 50 μl/well of 2,2azino-di(3-ethylbenzthiazoline)sulfonic acid (ABTS) (Zymed) in substratebuffer was then added. The plates were then read on a DynatechMicrotiter ELISA reader (410 nm) until maximum OD readings wereobtained. Mean OD readings were calculated and cICAM-1 concentrationswere calculated from the regression curve generated from the sICAM-1titration. The results from this ELISA for the sera from normal patientsand for sera from LAD patients, are presented in Table 1 below

                  TABLE I                                                         ______________________________________                                        cICAM-1 in Sera                                                               NORMAL HUMAN SERA (ng/ml)                                                                          LAD SERA (ng/ml)                                         ______________________________________                                        189                  335                                                      223                  687                                                      110                  372                                                      141                  363                                                      251                                                                            81                                                                           109                                                                           104                                                                           122                                                                           233                                                                           MEAN: 156            MEAN: 439                                                ______________________________________                                    

FIG. 2 is a graphic depiction of the cICAM-1 levels found using thisELISA in sera from normal patients (NHS), from LAD patients, and fromKawasakis patients.

These results indicate that cICAM-1 is present in serum and that itretains the functional epitope recognized by CL203.4 on domain 4 and thefunctional epitope recognized by R6.5 on domain 2.

D. Cell Adhesion Assay

50 μl of CA7 (10 μg/ml) was added to each well of 96 well flat bottomE.I.A. microtiter plates (Linbro) at room temperature for 1 hour. Wellswere then washed three times with DPBS and blocked with 200 μl of 2%BSA-DPBS for 1 hour at 37° C. 100 μl of normal human sera or 100 ng ofsICAM-1 in 1% BSA-DPBS were then added to each well and incubated for 1hour at 37° C. Wells were then washed three times with DPBS. 50 μl ofRPMI-1640 (Gibco, Grand Island, N.Y.) supplemented with 50 μg/ml ofgentamycin, 1 mM L-glutamine and 10% heat inactivated fetal bovine serum(Gibco) (complete medium), R3.1 (100 μg/ml), R6.5 (100 μg/ml), orCL203.4 (100 μg/ml) were then added to the appropriate wells. SKW-3cells (human T-lymphocyte cells from Sloan-Kettering Wallace,Sloan-Kettering Memorial, Rye, N.Y.) (50 μl of 2×10⁶ cells/ml) incomplete medium, were then added to each well and incubated for 30minutes at 37° C. The nonadherent cells were gently washed off withRPMI-1640. The number of adherent cells was determined by mitochondrialreduction of MTT (3[4,5-dimethylthiazol-2-yl]-22,5 diphenyl-tetrazoliumbromide:thiazole blue) (Sigma) as follows: The adherent cells wereincubated with 100 μl of complete medium and 20 μl of MTT (5 mg/ml) for3 hours at 37° C. The reduced MTT crystals so produced were solubilizedwith 60 μl/well of 1% Triton-X100 (Biorad, Richmond, Calif.) in 0.1NHCl. Plates were microwaved to gently heat the MTT precipitate. 10 μl ofethanol was added to remove any detergent bubbles. OD of the wells wasthen determined at 570 nM on a Dynatech Microtiter ELISA reader.

The above-described assay was repeated using the same procedure exceptthat: (1) no sICAM-1 and no human serum was added to the wells; (2) thewells were initially incubated with R6.5 instead of CA7; or (3) thewells were initially incubated with BSA instead of CA7.

The results of this assay are listed in Table 2 below.

                  TABLE 2                                                         ______________________________________                                        SKW-3 Adhesion                                                                               sICAM-1   sICAM-1                                                                              Serum   Serum                                 Trap  Block    MTT OD    % Inhib.                                                                             MTT OD  % Inhib.                              ______________________________________                                        CA7.sup.1                                                                           Media    427       --     458     --                                    CA7   R3.1     40        91     16      97                                    CA7   R6.5     95        75     137     70                                    CA7   CL203.4  417       3      503     0                                     R6.5  --       0         --     0       --                                    BSA   --       0         --     0       --                                    ______________________________________                                         .sup.1 Using CA7 as the trapping antibody in the absence of sICAM1 and        human serum resulted in no SKW3 cell adherence.                          

The results in Table 2 above demonstrate that cICAM-1 in human serumretains its capacity to mediate LFA-1 dependent adhesion. Binding ofSKW-3 cells were inhibited by both the anti-LFA-1 antibody (R3.1) andthe anti-ICAM-1 antibody which binds to domains 1 and 2 of ICAM-1(R6.5). These results indicate that cICAM-1 retains functional epitopesof domains 1, 2 and 5.

EXAMPLE 2 Detection of Pathological Pregnancy A. Preparation ofBiological Materials

sICAM-1 was prepared as described in Example 1A.

Amniotic fluid samples were obtained from the following clinical groups:(1) samples obtained by amniocentesis for genetic evaluation inassociation with advanced maternal age, at 16 weeks gestation (39); (2)samples obtained by amniocentesis for genetic evaluation following anelevated concentration of alpha-fetoprotein in the amniotic fluid (21);(3) samples obtained by amniocentesis for pulmonary maturity or atcesarean section with delivery prior to 37 weeks gestation (12); (4)samples obtained at cesarean section or by transcervical catherizationat the time of placement of fetal monitoring electrode, with delivery atterm (10).

Maternal serum alpha-fetoprotein concentrations were determined byradioimmunoassay. Elevated values were defined as >2.0 multiples of themedium for gestational age and used corrections for maternal weight andrace. In all cases in which maternal serum alpha-fetoproteinconcentrations were elevated, amniotic fluid alpha-fetoproteinconcentrations were normal.

B. Preparation of Monoclonal Antibodies

Monoclonal antibodies were prepared as described in Example 1B.

C. ELISA

The same assay as described in Example 1C was used except that theamniotic fluid samples were substituted for the sera samples.

The results of this ELISA are listed in Table 3 below.

                  TABLE 3                                                         ______________________________________                                        cICAM-1 Levels in Amniotic Fluid                                                                        cICAM-1 Level                                       Gestational Age                                                                             Total Samples                                                                             (ng/ml) ± SD                                     ______________________________________                                        16 Weeks      39          33 ± 8                                           Normal MSAFP.sup.1                                                            16 Weeks      21          131 ± 31                                         Elevated MSAFP.sup.1                                                          Term          5           192.5                                               Spontaneous Del.                                                              Term          4           140 ± 35                                         Cesaraen                                                                      ______________________________________                                         .sup.1 Maternal serum alphafetoprotein                                   

                  TABLE 4                                                         ______________________________________                                        cICAM-1 Levels in Preterm Deliveries                                                                    cICAM-1 Level                                       Condition     Total Samples                                                                             (ng/ml) ± SD                                     ______________________________________                                        Spontaneous Del.                                                                            5           718                                                 Other Delivery.sup.1                                                                        4           265                                                 Non-Inflamed.sup.2                                                                          10          336 ± 69                                         Inflamed.sup.2                                                                              2           1034                                                ______________________________________                                         .sup.1 Cesaraen                                                               .sup.2 Uterine compartment                                               

FIG. 3 is a graphic depiction of the average (mean) levels of cICAM-1 inthe amniotic fluid present at different gestational ages and conditions,determined using the ELISA described above.

These data show that a higher than normal level of cICAM-1 in theamniotic fluid prior to delivery at term, indicates a risk of anabnormal pregnancy.

EXAMPLE 3 Detection of cICAM-1 in Synovial Fluid of Arthritic PatientsA. Preparation of Biological Materials

sICAM-1 was prepared as described in Example 1A.

Samples of synovial fluid from patients suffering from rheumatoidarthritis (8), osteoarthritis (4), psoriatic arthritis (1), goutyarthritis (1) and chondrocalcinosis (1) were collected by arthroscopy.

B. Preparation of Monoclonal Antibodies

Monoclonal antibodies were prepared as described in Example 1B.

C. ELISA

cICAM-1 levels were measured using the ELISA described in Example 1C,substituting the samples of synovial fluid for the sera samples.

The results of this assay are graphically depicted in FIG. 4. Samplesfrom patients suffering from rheumatoid arthritis, which is accompaniedby inflammation, contained elevated levels of cICAM-1 whereas patientssuffering from osteoarthritis, which is not accompanied by inflammation,did not contain elevated levels of cICAM-1.

EXAMPLE 4 Detection of cICAM-1 in Bile of Liver Transplant Patients A.Preparation of Biological Materials

sICAM-1 was prepared as described in Example 1A.

Samples of bile and plasma from liver transplant patients who rejectedthe transplant (14), who had complications after the transplant (7) andwho were stable after the transplant (10), were collected each day for2-15 days after transplantation of the liver, as described in Adams etal, The Lancet, Mar. 4, 1989, pp. 469-471.

B. Preparation of Monoclonal Antibodies

Monoclonal antibodies were prepared as described in Example 1B.

C. ELISA

cICAM-1 levels in the bile samples were measured using the ELISAdescribed in Example 1C, substituting the bile samples for the serasamples.

FIG. 5 depicts the levels of cICAM-1 in the samples of bile and plasmaof a patient rejecting the transplant and in the samples of bile andplasma of a non-rejecting patient, over the course of 15 dayspost-transplant. Additional immunosuppressive therapy was institutedafter 11 days and resulted in a drop of cICAM-1 levels in the rejectingpatient.

FIG. 6 depicts the level of cICAM-1 measured in the samples of patientswho rejected the transplanted liver at the time of diagnosis ofrejection, the level of cICAM-1 measured in the samples of patients whoexperienced complications with the transplant, and the level of cICAM-1in the samples of stable patients. With the exemption of one sample, allof the samples of the patients who rejected the transplanted livercontained elevated levels of cICAM-1. The samples from the stable(non-rejecting) patients contained normal levels of cICAM-1.

EXAMPLE 5 Detection of cICAM-1 in Serum of Patients with Human MalignantMelanoma A. Preparation of Biological Materials

sICAM-1 was prepared as described in Example 1A.

Sera were obtained from patients with confirmed Stage I (14), Stage II(24) and Stage III (18) cutaneous malignant melanoma, and from 12 normalindividuals. Stage I disease was defined as the presence of a primarylesion with no clinically observable metastatic disease at the time ofserum collection; Stage II disease was defined as metastasis to regionallymph nodes, local recurrence with or without regional lymph nodeinvolvement, or initial presentation of melanoma in a single lymph nodegroup with no identifiable primary lesion; and Stage III disease wasremote cutanious, subcutaneous, or visceral metastases. Survival wasmeasured in months and was defined as the time from diagnosis to date ofevaluation or death.

B. Preparation of Monoclonal Antibodies

Monoclonal antibodies were prepared as described in Example 1B.

C. ELISA

cICAM-1 levels in the sera samples were measured using the ELISAdescribed in Example 1C.

The results of this assay are listed below in Table 5.

                  TABLE 5                                                         ______________________________________                                        cICAM-1 in Serum of Patients with Malignant Melanoma                                   cICAM-1                 % Positive                                   Patients (No.)                                                                         (Mean ± SEM.sup.1)                                                                        Range    Sera.sup.2                                   ______________________________________                                        Control (12)                                                                           166.0 ± 17.5                                                                              81-251   0    (0/12)                                  Stage I (14)                                                                           .sup. 406.2 ± 33.4).sup.3                                                                 265-661  93   (13/14)                                 Stage II (24)                                                                          232.9 ± 18.7                                                                              97-456   25   (6/24)                                  Stage III (18)                                                                         .sup. 303.8 ± 30.4.sup.4                                                                  78-493   60   (11/18)                                 ______________________________________                                         .sup.1 ng/ml of plasma (SEM = Standard Error of the Mean)                     .sup.2 Positive serum = >288.0 ng/ml                                          .sup.3 p <.001 when compared with control or Stage II patients                .sup.4 p <.01 when compared with control                                 

                  TABLE 6                                                         ______________________________________                                        Survival in Stage II and Stage III Melanoma Patients with                     Elevated Levels of cICAM-1                                                                          Survival.sup.2                                          Patients (No.)                                                                         cICAM-1.sup.1                                                                              (+SEM)      Range                                       ______________________________________                                        Stage II (6)                                                                           elevated     25.8 ± 5.4                                                                              7-41                                       Stage II (18)                                                                          normal       .sup. 38.8 ± 2.2.sup.3                                                                 11-50                                       Stage III (7)                                                                          elevated     33.6 ± 6.3                                                                             18-66                                       Stage III (6)                                                                          normal        64.3 ± 13.1.sup.4                                                                      35-120                                     ______________________________________                                         .sup.1 Level of serum cICAM1 in "elevated" positive patients >288.0 ng/ml     "normal" patients < 288.0 ng/ml                                               .sup.2 In months                                                              .sup.3 p <.01 when compared with Stage II "elevated                           .sup.4 p <.05 when compared with Stage III "elevated patients            

Serum levels of cICAM-1 were significantly elevated in patients withStage I and III melanoma, and markedly enhanced in Stage II patients.Abnormally high serum levels of cICAM-1 were present in 93% of Stage I,25% of Stage II, and 60% of Stage III patients, but were not observed innormal individuals.

Stage I patients, presumably with the least tumor burden, had bothsignificantly (p <0.001) increased levels of cICAM-1 and an extremelyhigh incidence (93%) of elevated levels of cICAM-1. All melanomapatients had unusually high levels of serum cICAM-1. In Stage IImelanoma patients with elevated levels of cICAM-1, a significantreduction in mean survival was observed when compared with Stage IIpatients with normal levels of cICAM-1. Mean survival was also reducedin Stage III patients with abnormally high levels of cICAM-1.

What is claimed is:
 1. A method for the detection of a pathological pregnancy in a pregnant female which comprises the steps of:a) contacting a sample of the amniotic fluid or the serum, from the pregnant female with an immobilized first antibody capable of binding to ICAM-1, the first immobilized antibody being insoluble in the sample, to form a first insoluble complex of cICAM-1 and the immobilized first antibody; b) contacting the first insoluble complex with a soluble labelled second antibody capable of binding to ICAM-1, to form a final insoluble complex of the labelled second antibody, cICAM-1 and the immobilized first antibody; c) separating the final insoluble complex from the sample and any unreacted soluble labelled second antibody; d) determining either the amount of label associated with the final insoluble complex or the amount of unreacted label, as a measure of the amount of cICAM-1 in the sample; and e) comparing the amount of cICAM-1 in the sample with standards of cICAM-1 normal for amniotic fluid or serum for the stage of pregnancy of the pregnant female and with higher than normal levels of cICAM-1 indicating a risk of an abnormal delivery.
 2. A method for the detection of a pathological pregnancy in a pregnant female which comprises measuring the amount of cICAM-1 is a sample of the amniotic fluid or the serum from the pregnant female, and then comparing the amount of cICAM-1 in the sample to standards of cICAM-1 normal for amniotic fluid or serum for the stage of pregnancy of the pregnant female and with higher than normal levels of cICAM-1 indicating a risk of an abnormal delivery.
 3. A method for the detection of a pathological pregnancy in a pregnant female which comprises the steps of:a) contacting a sample of the amniotic fluid or the serum of the pregnant female, with a first antibody capable of binding to ICAM-1 and labelled second antibody cable of binding to ICAM-1; b) determining the amount of bound labelled second antibody as a measure of the amount of cICAM-1 in the sample; and c) comparing the amount of cICAM-1 in the sample with standards of cICAM-1 normal for amniotic fluid or serum for the stage of pregnancy of the pregnant female for the stage of pregnancy of the pregnant female and with higher than normal levels of cICAM-1 indicating a risk of an abnormal delivery.
 4. A method as recited in claim 3 wherein the first antibody and the labelled second antibody are monoclonal antibodies.
 5. A method as recited in claim 4 wherein the first antibody is immobilized and the labelled second antibody is soluble.
 6. A method as recited in claim 5 wherein the immobilized first antibody is immobilized on a solid support. 